AGEs promote calcification of HASMCs by mediating Pi3k/AKT-GSK3ß signaling.

This study aimed to investigate the effects of advanced glycation end products (AGEs) on the calcification of human arterial smooth muscle cells (HASMCs) and to explore whether AGEs can promote the calcification of HASMCs by activating the phosphoinositide 3-kinase (PI3K)/AKT-glycogen synthase kinase 3 beta (GSK3-ß) axis. Cultured HASMCs were divided into five groups: blank control group, dimethyl sulfoxide (vehicle) group, AGEs group, LY294002 (AKT inhibitor) group, and TWS119 (GSK3-ß inhibitor) group. Cells were pretreated with either vehicle, LY294002, or TWS119 for 2 hours followed by incubation with AGEs (25 µg/mL) for 5 days, and the expression levels of proteins in each group were analyzed by western blotting. AGE treatment promoted HASMC calcification, which coincided with increased expression of p-AKT and p-GSK3-ß (serine 9). Also, AGEs upregulated the expression of osteoprotegerin and bone morphogenetic protein, and these effects were suppressed by LY294002 but enhanced by TWS119. In conclusion, AGEs promote calcification of HASMCs, and this effect is ameliorated by inhibition of AKT activity but potentiated by inhibition of GSK3-ß activity. Hence, AGEs trigger HASMC calcification by regulating PI3K/AKT-GSK3-ß signaling.

[Towards Understanding the Pathophysiological Significance of Cytokine Trapping Mediated by Advanced Glycation End Products].

Advanced glycation end products (AGEs) are non-enzymatically formed from sugars or their metabolites with biomolecules. These molecules are formed in vivo, and the formation of AGEs on functional biomolecules was demonstrated to alter their properties. In addition, AGEs were reported to elicit inflammatory reactions by stimulating their endogenous receptors. However, the relationship between AGEs and these phenomena remains unclear. To understand the pathophysiological roles of AGEs, we investigated their action mechanisms at the molecular level. In this study, we found that AGEs can directly interact with tumor necrosis factor-like weak inducer of apoptosis (TWEAK), the cytokine that controls tumor necrosis factor-α (TNF-α)-stimulated inflammatory reactions. This interaction inhibited the action of TWEAK and subsequently increased TNF-α-induced proinflammatory cytokine expression. This raised the possibility that AGEs trap other cytokines and alter their activities. We named this hypothesis "AGE-mediated cytokine trapping". To assess this hypothesis, we next examined the mechanism of AGE-TWEAK interaction. The pull-down assay using the deletion mutant revealed that a relatively large region of TWEAK functions in the interaction with AGEs, suggesting that it is difficult to explore other cytokines capable of interacting with AGEs using TWEAK sequence similarity. Therefore, to find novel AGE-cytokine interactions, we performed comprehensive screening using a protein array and found several candidates. To generalize "AGE-mediated cytokine trapping", detailed studies using these candidates are now in progress.

Advanced glycation end products reduce macrophage-mediated killing of Staphylococcus aureus by ARL8 upregulation and inhibition of autolysosome formation.

Staphylococcus aureus, a pathogen most frequently found in diabetic foot ulcer infection, was recently suggested as an intracellular pathogen. Autophagy in professional phagocytes like macrophages allows selective destruction of intracellular pathogens, and its dysfunction can increase the survival of internalized pathogens, causing infections to worsen and spread. Previous works have shown that S. aureus infections in diabetes appeared more severe and invasive, and coincided with the suppressed autophagy in dermal tissues of diabetic rat, but the exact mechanisms are unclear. Here, we demonstrated that accumulation of advanced glycation end products (AGEs) contributed to the diminished autophagy-mediated clearance of S. aureus in the macrophages differentiated from PMA-treated human monocytic cell line THP-1. Importantly, infected macrophages showed increased S. aureus containing autophagosome, but the subsequent fusion of S. aureus containing autophagosome and lysosome was suppressed in AGEs-pretreated cells, suggesting AGEs blocked the autophagic flux and enabled S. aureus survival and escape. At the molecular level, elevated lysosomal ARL8 expression in AGEs-treated macrophages was required for AGEs-mediated inhibition of autophagosome-lysosome fusion. Silencing ARL8 in AGEs-treated macrophages restored autophagic flux and increased S. aureus clearance. Our results therefore demonstrate a new mechanism, in which AGEs accelerate S. aureus immune evasion in macrophages by ARL8-dependent suppression of autophagosome-lysosome fusion and bactericidal capability.

Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells.

Purpose: We used a human corneal epithelial cell (HCE) line to determine the involvement of the advanced glycation end products (AGEs) / receptor for AGEs (RAGE) couple in corneal epithelium wound healing. Methods: After wounding, HCE cells were exposed to two major RAGE ligands (HMGB1 and AGEs), and wound healing was evaluated using the in vitro scratch assay. Following wound healing, the HCE cells were used to study the influence of the RAGE ligands on HCE proliferation, invasion, and migration. Activation of the nuclear factor (NF)-κB signaling pathway by the AGEs/RAGE couple was tested using a luciferase reporter assay. Functional transcriptional regulation by this pathway was confirmed by quantification of expression of the connexin 43 target gene. For each experiment, specific RAGE involvement was confirmed by small interfering RNA treatments. Results: AGEs treatment at a dose of 100 µg/mL significantly improved the wound healing process in a RAGE-dependent manner by promoting cell migration, whereas HMGB1 had no effect. No significant influence of the AGEs/RAGE couple was observed on cell proliferation and invasion. However, this treatment induced an early activation of the NF-κB pathway and positively regulated the expression of the target gene, connexin 43, at both the mRNA and protein levels. Conclusions: Our results demonstrate that the RAGE pathway is activated by AGEs treatment and is involved in the promotion of corneal epithelial wound healing. This positive action is observed only during the early stages of wound healing, as illustrated by the quick activation of the NF-κB pathway and induction of connexin 43 expression.

Identification of a novel advanced glycation end product derived from lactaldehyde.

Advanced glycation end products (AGEs) are implicated in the development of diabetic complications via the receptor for AGEs (RAGE). We have reported that the 3-hydroxypyridinium (3HP)-containing AGEs derived from α-hydroxyaldehydes physically interact with RAGE and show cytotoxicity. Lactaldehyde (LA) is formed from a reaction between threonine and myeloperoxidase, but no LA-derived AGEs have been characterized. Here, we identify the structure and physiological effects of an AGE derived from LA. We isolated a novel 3HP derivative, 2-acetamido-6-(3-hydroxy-5-methyl-pyridin-1-ium-1-yl)hexanoate, named as N-acetyl-LAPL (lactaldehyde-derived pyridinium-type lysine adduct), from a mixture of LA with Nα-acetyl-L-lysine. LAPL was also detected in the LA-modified protein. LAPL elicited toxicity in PC12 neuronal cells, but the effect was suppressed by the soluble form of RAGE as a decoy receptor. Moreover, surface plasmon resonance-based analysis revealed that LAPL specifically binds to recombinant RAGE. These results indicate that LA generates an AGE containing the 3HP moiety and contributes to RAGE-dependent cytotoxicity. Abbreviations: AGEs: advanced glycation end products; RAGE: receptor for advanced glycation end products; 3HP: 3-hydroxypyridinium; LA: lactaldehyde; LAPL: lactaldehyde-derived pyridinium-type lysine adduct; BSA: bovine serum albumin; GLAP: glyceraldehyde-derived pyridinium; MPO: myeloperoxidase; HFBA: heptafluorobutyric acid; TFA: trifluoroacetic acid; HPLC: high performance liquid chromatography; LC-ESI-QTOF-MS: liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry; NMR: nuclear magnetic resonance; LA-BSA: lactaldehyde-modified bovine serum albumin; PBS: phosphate buffered saline, GST, glutathione S-transferase; SPR: surface plasmon resonance; OP-lysine: 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate; GLO1: glyoxalase 1; MG, methylglyoxal.

Gene-disease associations identify a connectome with shared molecular pathways in human cholangiopathies.

Cholangiopathies are a diverse group of progressive diseases whose primary cell targets are cholangiocytes. To identify shared pathogenesis and molecular connectivity among the three main human cholangiopathies (biliary atresia [BA], primary biliary cholangitis [PBC], and primary sclerosing cholangitis [PSC]), we built a comprehensive platform of published data on gene variants, gene expression, and functional studies and applied network-based analytics in the search for shared molecular circuits. Mining the data platform with largest connected component and interactome analyses, we validated previously reported associations and identified essential and hub genes. In addition to disease-specific modules, we found a substantial overlap of disease neighborhoods and uncovered a group of 34 core genes that are enriched for immune processes and abnormal intestine/hepatobiliary mouse phenotypes. Within this core, we identified a gene subcore containing signal transduction and activator of transcription 3, interleukin-6, tumor necrosis factor, and forkhead box P3 prominently placed in a regulatory connectome of genes related to cellular immunity and fibrosis. We also found substantial gene enrichment in the advanced glycation endproduct/receptor for advanced glycation endproducts (RAGE) pathway and showed that RAGE activation induced cholangiocyte proliferation. Conclusion: Human cholangiopathies share pathways enriched by immunity genes and a molecular connectome that links different pathogenic features of BA, PBC, and PSC. (Hepatology 2018;67:676-689).

Polycystic ovarian syndrome-associated cardiovascular complications: An overview of the association between the biochemical markers and potential strategies for their prevention and elimination.

Polycystic ovarian syndrome (PCOS) is associated with multiple cardiovascular risk factors (CVRF) including endothelial dysfunction (ED) and presence of metabolic syndrome (MS). The probable reason suggested for elevated CVRF in PCOS is oxidative stress (OS), which is an integral factor in cardiometabolic complications (CMC) seen in PCOS women. The interrelated mechanisms by which CVRF instigate clinical manifestation plays a crucial role in identification of a strategy to treat different comorbidities in PCOS. The existing treatment for PCOS mostly focuses on management of individual disorders, however, therapeutic strategies or novel targets to address cardiovascular complications in PCOS deserve extensive analysis.

Advanced glycation end products attenuate the function of tumor necrosis factor-like weak inducer of apoptosis to regulate the inflammatory response.

Advanced glycation end products (AGEs) are formed from the non-enzymatic glycation reaction of reducing sugars or their metabolites with the free amino groups of several biomolecules and are known to play pathophysiological roles in various inflammatory diseases. In an earlier study, it was suggested that tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has a unique role to regulate the tumor necrosis factor α (TNFα)-induced inflammatory response. In this study, we investigated the effect of the AGEs-TWEAK interaction on proinflammatory signaling responses in endothelial cells and the influence of AGEs on the cellular function of TWEAK in the inflammatory process. The effect of AGEs on the TWEAK/TNFα-induced gene expression of interleukin-8 (IL-8) was determined by real-time RT-PCR in endothelial-like EA.hy.926 cells. The pull-down assay was performed using recombinant His-tagged TWEAK and AGEs. The NF-κB activation was analyzed by Western blotting with canonical and non-canonical pathway-specific antibodies. AGEs dose-dependently inhibited TWEAK-induced IL-8 gene expression, whereas AGEs themselves had almost no effect on IL-8 expression. AGEs were found to bind directly to TWEAK in the pull-down assay. TNFα-induced IL-8 production and canonical NF-κB activation were suppressed by TWEAK pretreatment, whereas TWEAK-induced non-canonical NF-κB activation was enhanced by pretreatment. These effects induced by TWEAK pretreatment were abolished by the co-addition of AGEs. Our findings suggest that AGEs attenuate the function of TWEAK to regulate the TNFα-induced inflammatory responses, which provide important clues for understanding the significance of the AGEs-TWEAK interaction in inflammatory processes.

Inhibition of protein glycation, antioxidant and antiproliferative activities of Carpobrotus edulis extracts.

Carpobrotus edulis is an important South African medicinal plants used as a food and therapeutic agent in traditional medicine. The aim of this study was to determine the phytochemical content, antioxidant, antiglycation and cytotoxic effect against Human Colon Cancer Cell Line (HCT-116) of aqueous and ethanol-water (1:1v/v) extracts of Carpobrotus edulis.The content of total phenolics and flavonoids in aqueous and ethanol-water extract were 151.99µg and 66.35µg gallic acid equivalents/mg of dry extract, and 38.84µg and 21.96µg quercetin/mg of dry extract, respectively. Furthermore, phenolic compositions analysis indicated the presence of seven majority compounds including sinapic acid, ferulic acid, luteolin7-o-glucoside, hyperoside, isoquercitrin, ellagic acid and isorhamnetin 3-O-rutinoside. The ethanol-water extract (100-1000µg/mL) showed better antioxidant activity than aqueous extract. Furthermore, Carpobrotus edulis extracts, especially ethanol-water extract significantly inhibited the formation of fluorescent advanced glycation end products, prevented oxidation-induced protein damage and exhibited a cytotoxic effect against HCT116 cells, with a significant decrease in cell viability after 24h of incubation. The results obtained suggest that the Carpobrotus edulis extracts could be used as an easily accessible source of natural antioxidants and as potential phytochemicals against protein glycation and colon cancer.

Downregulation of miR-146a, cyclooxygenase-2 and advanced glycation end-products in simvastatin-treated older patients with hyperlipidemia.

AIM: Hyperlipidemia is a disease with abnormally elevated levels of lipids/lipoproteins in the blood, and it is regarded as an important risk factor for cardiovascular and cerebrovascular diseases. Statins have been found to prevent vascular diseases by reducing low-density lipoprotein cholesterol and regulation of immune responses. Here, we aim to study the expression change of immune-related microRNA and genes in older patients with hyperlipidemia after treatment with simvastatin. METHODS: A total of 25 older male patients with hyperlipidemia were included in the study and received simvastatin treatment (20 mg/day). Clinical characteristics of these patients were examined, including lipoprotein cholesterol, high-sensitivity C-reactive protein, blood routine and biochemical characters. We tested miR-146a, interleukin-1-receptor-associated kinase 1, tumor necrosis factor-receptor-associated factor 6 and cyclooxygenase-2 level by real-time polymerase chain reaction, and expressions of advanced glycation end-products, p53 and p21 were analyzed by enzyme-linked immunosorbent assay. RESULTS: Simvastatin treatment effectively reduced total cholesterol and low-density lipoprotein cholesterol, but had little effect on high-density lipoprotein cholesterol. High-sensitivity C-reactive protein was slightly reduced. Expression of cyclooxygenase-2 and advanced glycation end-products were significantly reduced. Furthermore, simvastatin effectively reduced the expression of p53 and p21. Significantly downregulated miR-146a, and an obvious reduction of interleukin-1-receptor-associated kinase 1 were also detected, whereas tumor necrosis factor-receptor-associated factor 6 remained unchanged. Besides, there was a significant reduction of alanine transaminase, aspertate aminotransferase, alkaline phosphatase and lactate dehydrogenase. CONCLUSION: Simvastatin treatment could inhibit inflammation and senescence-associated genes in older patients with hyperlipidemia, suggesting its application in inflammatory and age-related diseases.

Advanced glycation end-products and insulin signaling in granulosa cells.

Advanced glycation end-products (AGEs) may interfere with insulin intracellular signaling and glucose transport in human granulosa cells, potentially affecting ovarian function, follicular growth, linked with diminished fertility. The potential interaction of AGEs with insulin signaling pathways and glucose transport was investigated in human granulosa KGN cells. KGN cells were cultured with variable concentrations of human glycated albumin (HGA, 50-200 µg/mL) or insulin (100 ng/mL). Combined treatments of KGN cells with insulin (100 ng/mL) and HGA (200 µg/mL) were also performed. p-AKT levels and glucose transporter type 4 (Glut-4) translocation analysis were performed by Western blot. Phosphatidylinositol-3-kinase (PI3K)-specific signaling was checked by using the PI3K-inhibitor, LY294002. p-AKT levels were significantly increased following insulin treatment compared to basal levels or HGA exposure. This insulin-mediated AKT-phosphorylation was PI3K-specific and it was inhibited after combined treatment of insulin and HGA. Furthermore, Glut-4 translocation from the cytoplasm to the membrane compartments of KGN cells was remarkably reduced after the combined treatment of insulin and HGA. The present findings support that AGEs interfere with insulin signaling in granulosa cells and prevent Glut-4 membrane translocation suggesting that intra ovarian AGEs accumulation, from endogenous or exogenous sources, may contribute to the pathophysiology of states characterized with anovulation and insulin resistance such as polycystic ovary syndrome.

Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway.

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.

OSSC1E-K19, a novel phytochemical component of Osteomeles schwerinae, prevents glycated albumin-induced retinal vascular injury in rats.

In the pathophysiology of diabetic retinopathy (DR), advanced glycation end products (AGEs) and vascular endothelial growth factor (VEGF) are thought to have important roles. It is known that VEGF causes a breakdown of the blood­retinal barrier (BRB) and retinal neovascularization; however, how AGEs affect the retina has largely remained elusive. OSSC1E­K19 is a novel phytochemical component of Osteomeles schwerinae. The objective of the present study was to evaluate the protective effects of OSSC1E­K19 on retinal vascular injury in AGE­modified rat serum albumin (AGE-RSA)-induced retinopathy. AGE-RSA-injected rat eyes were used investigate the protective effects of OSSC1E­K19 on BRB breakdown. Intravitreal injection of OSSC1E-K19 prevented AGE-RSA-induced BRB breakdown and decreased retinal VEGF expression in retinal vessels. In addition, OSSC1E-K19 inhibited the loss of occludin, a significant tight junction protein. These results supported the potential therapeutic utility of OSSC1E-K19 for retinal vascular permeability diseases.

Role of receptor for advanced glycation end products in the complication and progression of various types of cancers.

BACKGROUND: Receptor for advanced glycation end-products popularly known as RAGE is a cell surface immunoglobulin class of molecule, binds with multiple ligands and therefore considered as a multi-ligand receptor. Use of RAGE deficient mice (RAGE(-/-)) as well as established mouse models pertaining to inflammation-associated carcinogenesis such as that of chemically induced carcinogenesis and colitis associated cancer provides a direct genetic evidence for a likelihood novel role of RAGE in cancer, with respect to its ability to lead cancer cell proliferation and survival. Besides inflammation, interaction of RAGE with its various ligands enhances oxidative stress both in cancerous and noncancerous cells which further complicates the progression of cancers. SCOPE OF REVIEW: Till date, no single review article has discussed the mechanism of RAGE dependent complication of cancers, particularly the role of RAGE in cancer cell proliferation, angiogenesis, survival and anti-apoptosis needs to be discussed. MAJOR CONCLUSION: RAGE enhances the number of cancer cells by activating the cell cycle proteins (e.g., cyclin D1), anti-apoptotic proteins (e.g., BCl2), prosurvival (AKT) and autophagic proteins. Role of RAGE has also been detected in formation of new blood vessels (angiogenesis) in the cancer cells and activation of myeloid derived suppressor cells (MDSCs). GENERAL SIGNIFICANCE: This review article describes the role of RAGE in the complication of various types of cancers and the possible usefulness of RAGE dependent therapy to confront cancers in a stronger magnitude.

Oligonol, a low-molecular-weight polyphenol derived from lychee fruit, attenuates gluco-lipotoxicity-mediated renal disorder in type 2 diabetic db/db mice.

Oligonol is a phenolic product derived from lychee fruit extract containing catechin-type monomers and oligomers of proanthocyanidins, produced by a manufacturing process which converts polyphenol polymers into oligomers. These proanthocyanidins have been reported to exhibit beneficial bioactivities in many studies, and so oligonol, a rich source of polyphenol, is expected to show favorable effects on various chronic diseases. This article summarizes recent work whether oligonol has an ameliorative effect on diabetic indices and renal disorders associated with gluco-lipotoxicity-mediated oxidative stress, inflammation, and apoptosis in db/db mice with diabetes. Oligonol was able to improve diabetic indices, prevent the development of diabetic renal disease, and preserve renal cells and the renal morphological structure via the attenuation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-induced oxidative stress, inhibition of advanced glycation endproduct (AGE) generation, and prevention of apoptosis-induced cell death in db/db mice, being independent of changes in the body weight or serum glucose levels. The present study provides important evidence that oligonol exhibits a pleiotropic effect, representing renoprotective effects against the development of diabetic complications in type 2 diabetic db/db mice.

Amyloid-beta mediates the receptor of advanced glycation end product-induced pro-inflammatory response via toll-like receptor 4 signaling pathway in retinal ganglion cell line RGC-5.

Patients with diabetes mellitus have an increased risk of developing Alzheimer's disease. Amyloid-ß, a product of amyloid precursor protein, is associated with neuro-inflammation in patients with Alzheimer's diseases. The correlation between amyloid-beta and advanced glycation end products, which accumulate in tissue of diabetic patients, is not clear. The aims of this study were to determine the effect of advanced glycation end product on the expression of amyloid precursor protein/amyloid-beta and associated pro-inflammatory responses in retinal ganglion cell line RGC-5. Treatment with advanced glycation end product produced upregulation of amyloid precursor protein and increased secretion of amyloid-ß(1-40). Additionally, amyloid-ß(1-40) induced toll-like receptor 4-dependent phosphorylation of tyrosine in myeloid differentiation primary response gene (88). We found that N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a γ-secretase inhibitor, reduced the secretion of amyloid-ß(1-40) and inhibited the advanced glycation end product-induced activation of myeloid differentiation primary response gene (88). Amyloid-ß(1-40) induced the activation of NF-κB and the expression of TNFα mRNA. Knockdown of toll-like receptor 4 inhibited the amyloid-ß(1-40)-induced phosphorylation of p65 in NF-κB. Additionally, the nuclear translocation of p65 and transcriptions of TNFα were inhibited by siRNA knockdown of receptor of advanced glycation end product or toll-like receptor 4. The advanced glycation end product-induced secretion of VEGF-A was also reduced by knockdown of toll-like receptor 4. Taken together, our data suggested that amyloid-ß(1-40) mediates the interaction between receptor of advanced glycation end product and toll-like receptor 4. Inhibition of the toll-like receptor 4 is an effective method for suppressing the amyloid-ß(1-40)-induced pro-inflammatory responses in RGC-5 cells.

DHA inhibited AGEs-induced retinal microglia activation via suppression of the PPARγ/NFκB pathway and reduction of signal transducers in the AGEs/RAGE axis recruitment into lipid rafts.

Recent studies revealed that dietary intake of docosahexaenoic acid (DHA) prevented diabetic retinopathy (DR), but the underlying mechanism was not fully understood. Retinal microglia are a specialized population of macrophages in retina. Considerable evidence has shown that microglia activation may trigger neuronal death and vascular dysfunction in DR. The aim of this study was to investigate the effects of DHA on advanced glycation end products (AGEs)-induced microglia activation using an in vitro microglia culture system, and concurrently to explore the mediating mechanisms. DHA inhibited AGEs-induced microglia activation and tumor necrosis factor α (TNFα) secretion. These effects of DHA were directly linked with suppression of nuclear factor-kappa B (NFκB) activity, as evident by the reduction of p-IκBα expression, p-NFκB p65 nucleus translocation, NFκB DNA binding activity, and the regulation of gene transcription (TNFα, IL-1ß, ICAM-1, and RAGE mRNA). Furthermore, DHA significantly increased phosphorylation of peroxisome proliferator-activated receptor-gamma (PPARγ), and combined with PPARγ stealth RNAi oligonucleotide, we confirmed that DHA inhibition of AGEs-induced microglia activation was partially through the PPARγ/NFκB pathway. Moreover, although AGEs incubation dramatically elevated expression of the cell surface receptor for AGEs (RAGE), DHA significantly inhibited RAGE and Src recruitment into lipid rafts. The AGEs-RAGE axis downstream signal transducers increased mitogen-activated protein kinase (p38 and JNK) phosphorylation. Taken together, DHA might inhibit AGEs-induced microglia activation via suppression of the PPARγ/NFκB pathway, and reduction of RAGE and AGEs/RAGE transducer recruitment into lipid rafts. These results provide a novel potential mechanism for the anti-inflammatory effects of DHA in DR prevention.

Advanced glycation end products are mitogenic signals and trigger cell cycle reentry of neurons in Alzheimer's disease brain.

Neurons that reenter the cell cycle die rather than divide, a phenomenon that is associated with neurodegeneration in Alzheimer's disease (AD). Reexpression of cell-cycle related genes in differentiated neurons in AD might be rooted in aberrant mitogenic signaling. Because microglia and astroglia proliferate in the vicinity of amyloid plaques, it is likely that plaque components or factors secreted from plaque-activated glia induce neuronal mitogenic signaling. Advanced glycation end products (AGEs), protein-bound oxidation products of sugar, might be one of those mitogenic compounds. Cyclin D1 positive neurons are colocalized with AGEs or directly surrounded by extracellular AGE deposits in AD brain. However, a direct proof of DNA replication in these cells has been missing. Here, we report by using fluorescent in situ hybridization that consistent with the expression of cell cycle proteins, hyperploid neuronal cells are in colocalization with AGE staining in AD brains but not in nondemented controls. To complement human data, we used apolipoprotein E-deficient mice as model of neurodegeneration and showed that increased oxidative stress caused an intensified neuronal deposition of AGEs, being accompanied by an activation of the MAPK cascade via RAGE. This cascade, in turn, induced the expression of cyclin D1 and DNA replication. In addition, reduction of oxidative stress by application of α-lipoic acid decreased AGE accumulations, and this decrease was accompanied by a reduction in cell cycle reentry and a more euploid neuronal genome.

Etomidate deteriorates the toxicity of advanced glycation end products to human endothelial Eahy926 cells.

Patients with diabetes mellitus, particularly those with cardiovascular complications, have increased risk of mortality when subject to anesthetics and surgery, compared with non-diabetic patients. Anesthetics may exert pressure on the cardiovascular system of diabetic patients, directly or by aggravating pre-existing cardiovascular complications. Advanced glycation end products (AGEs) are extremely accumulated in diabetes mellitus, and are confirmed to play an important role in the pathogenesis of diabetic microvascular and macrovascular complications. The purpose of the present study was to investigate the regulatory role of etomidate, which is widely used as intravenous general anesthetics, on the viability and apoptosis of human endothelial Eahy926 cells, in the presence of AGEs. The results demonstrated that etomidate and Glu-BSA (one type of AGE) synergistically reduced the human endothelial Eahy926 cell viability and induced cell apoptosis. In addition, western blot assay of apoptosis-associated molecules indicated that both agents synergistically upregulated the cytochrome c release, activated the apoptosis executor, caspase 3, and promoted the poly-ADP-ribose polymerase (PARP) lysis. Further results confirmed that the two agents synergistically promoted oxidative stress by decreasing mitochondrial respiratory chain complex IV and mitochondrial membrane potential (MMP), while upregulating reactive oxygen species (ROS) and mitochondrial superoxide. In conclusion, the results presented in this study offer a novel insight into the mechanisms of endothelial cell apoptosis in response to etomidate in the presence of AGEs. These results suggest that oxidative stress has important role in the synergistic promotion of apoptosis by etomidate and AGEs in endothelial Eahy926 cells.

Diabetes mellitus and ischemic diseases: molecular mechanisms of vascular repair dysfunction.

In patients with diabetes mellitus, the ability of ischemic tissue to synchronize the molecular and cellular events leading to restoration of tissue perfusion in response to the atherosclerotic occlusion of a patent artery is markedly impaired. As a consequence, adverse tissue remodeling and the extent of ischemic injury are intensified, leading to increased morbidity and mortality. Growing evidence from preclinical and clinical studies has implicated alterations in hypoxia-inducible factor 1 levels in the abrogation of proangiogenic pathways, including vascular endothelial growth factor A/phosphoinositide 3' kinase/AKT/endothelial nitric oxide synthase and in the activation of antiangiogenic signals characterized by accumulation of advanced glycation end products, reactive oxygen species overproduction, and endoplasmic reticulum stress. In addition, the diabetic milieu shows a switch toward proinflammatory antiregenerative pathways. Finally, the mobilization, subsequent recruitment, and the proangiogenic potential of the different subsets of angiogenesis-promoting bone marrow-derived cells are markedly impaired in the diabetic environment. In this review, we will give an overview of the current understanding on the signaling molecules contributing to the diabetes mellitus-induced impairment of postischemic revascularization mainly in the setting of myocardial infarction or critical limb ischemia.